Millions of couples in the United States are affected by infertility. In a large fraction of these couples, the infertility originates from a male defect that is associated not only with oligozoospermia but also with asthenozoospermia. Spermatozoa acquire their motility and capacity to fertilize as they traverse the epididymis, but our knowledge of the contribution of post-testicular mechanisms to these critical sperm functions is poor. The lumen of the epididymis and vas deferens (VD) is maintained at an acidic pH and has a low bicarbonate concentration: both are important for sperm maturation and for the maintenance of sperm in a quiescent state during their storage period. Thus, defective epididymal acidification might impair male reproductive capacity. Luminal acidification involves a concerted interaction between the different cell types that constitute the epididymal epithelium, mainly the clear and principal cells. We plan to dissect the factors involved in the establishment and maintenance of the mature phenotypes of these cells, as well as factors that coordinate the acid/base transport activities of these differentiated cells. SPECIFIC AIM 1 will examine cell-specific patterns of mRNA and protein expression in the epididymis during: A) post-natal development;and B) hormonal treatment or orchidectomy of adult animals. We will use our novel transgenic (Bl-EGFP) mice, in which enhanced green fluorescent protein (EGFP) is expressed specifically in narrow and clear cells. Selected clear and principal cells will be harvested by fluorescence assisted laser capture microdissection (FLCM), and mRNA expression will be examined by real-time PCR. Selected genes related to acid/base transport (H+ATPase, CAII, NHE3, NBC, etc.) and its regulation (PLC, PKA, PKC, gelsolin, etc) will be studied. Selected genes involved in epithelial cell differentiation, cell shape determination and the retention of specific cell types within the epithelium (cytoskeletal proteins, extracellular matrix proteins and cell adhesion molecules, proteins involved in apoptosis) will also be examined. Protein expression will be analyzed by immunofluorescence and Western blotting. SPECIFIC AIM 2 will characterize cell specific signaling pathways in the epididymis. A) As a sensitive real-time marker of activation of key cell-specific responses to stimuli (e.g. receptor activation leading to H+ATPase exocytosis), intracellular calcium will be measured by spectrofluorometry in positively identified clear and principal cells from our Bl-EGFP mice using Fura-2. B) We will also use a novel assay, developed using these mice, to follow in real time, cell shape variations in individual living clear cells, following activation or inhibition of proton secretion. These studies will help to elucidate key biological mechanisms that establish the proper luminal acid/base balance that leads to the optimum environment for sperm maturation and storage.